Zonal electrophoresis, and particularly gel electrophoresis is one of the best known methods for separation, purification and characterization of charged molecules, particularly macromolecules such as proteins or nucleic acids (Freifelder, Physical Biochemistry, 2nd ed., (1982) pp. 276-310, Freeman, San Franciso). Electrophoresis can be used to separate molecules based on their size, charge, conformation, and many combinations of these properties.
In most electrophoresis applications, charged molecules migrate through a supporting medium under the influence of an electric field. The supporting medium acts to suppress convection and diffusion, and can be sieving or nonsieving. In affinity electrophoresis, the support medium is also modified with chemical groups (hereinafter "capture ligands") that interact specifically or nonspecifically with one or more desired targets and, thus, help to accomplish the separation of target and non-target sample components during purification by influencing the mobility of the target.
Affinity electrophoresis has been used to enhance the separtion of nucleic acids. For example, Inami, et al. demonstrated that A:T-rich sequences of nucleic acids have an affinity for malechite green copolymerized with acrylamide and immobilized within an agarose gel (Inami, et al., Nucleic Acids Symp. Ser. (19 ) 29:77-8). In addition, vinyl-adenine modified polyacrylmide media has been used to resolve nucleic acids in capillary electrophoresis (Baba et al., Analytical Chemisty (1992), 64:1920-1924).
Electrophoresis techniques have been used by biochemist to study melting transitions. Electrophoretic mobility is a very sensitive indicator of protein and nucleic acid conformation and, therefore, electrophoresis is a very useful or studying melting induced by chemical or physical denaturants. Gels containing gradients of urea have been used to study the binding of proteins (Creighton, J Mol. Biol. (1979) 129:253-264) and melting behavior of nucleic acids (Fischer, et al., Cell (1979)16:191-200).
Temperature gradients have also been used to study these properties with similar results (Thatcher, et al., Biochem. J (1981), 197:105-109), and many methods of producing temperature gradients have been developed (Henco, et al., Methods Mol. Biol. (1994) 31:211-28 and Rosenbaum, et al., Biophys. Chem. (1987) 26:235-246).
While many advances have been made in the resolving power of electrophoresis, nucleic acids that contain only slight structural differences, for example, one or more point mutation in a long nucleic acid sequence, still cannot be successfully separated.
Analytical techniques that improve resolution of biological molecules are needed to provide researchers with the opportunity to further probe and understand biological systems.